Cytopathology

Cytopathology, often called cytology, is the study of pathologic changes in cells.

It is often divided into gynecologic and non-gynecologic. Gynecologic in this context usually refers to Pap test specimens, i.e. uterine cervix, vaginal vault; other gynecologic specimens are considered non-gynecologic.

Slide-marking conventions

Conventions are important for facilitating communication between various team members.

Common conventions:

• The slide is placed with the label on the right hand side.
• "Marks-of-interest", e.g. dots, are place above the field of interest (when viewed through a microscope - that does not correct for image inversion).
  • Microscopes used for surgical pathology, generally, do not correct for image inversion.
    • The "marks-of-interest" appear below the field of interest when the slide is viewed with the naked eye.

Marks-of-interest:

• Dot - region of interest.
• Inverted "V" - transformation zone (in cervical cytology specimen).
• Inverted "Y" - benign finding.

Notes:

• Image inversion in the microscope comes about as light travels in a straight line.
• Marks should draw attention to the best examples of the highest grade lesion present.
  • More than 7 marks on slide is generally considered excessive.

Dictums of cytopathology

• Malignant cells are malignant because they have malignant features.
• Benign cells are benign because they lack malignant features and have benign features.

Corollaries:

• Cells lacking malignant features are not malignant.
• Cells lacking benign features are not malignant.

Cytologic features of malignancy

Cytologic features of malignancy:

1. High nuclear-to-cytoplasmic ratio.
2. Nuclear membrane irregularities.
   • Notches/sharp discontinuities.
3. Chromatin abnormalities.
   • Clumped chromatin, esp. differences between quadrants of a nucleus.
   • Chromatin clumping at the nuclear membrane (chromatin margination).
4. Hyperchromatic nucleus (when compared to other cells).
5. Nuclear pleomorphism (comparison of neighbouring cells).
   • Def'n: variable staining, shape, and size.

Notes:

• Not all features of malignancy will be present in one cell.

Cell types

WG's cell typing guide:

	Nucleus location	Nucleus shape	Cytoplasm	Chromatin	Nucleoli	Other	Architecture
Squamous	Center	Oval/Spindle	Solid/dense	Coarse	Absent	Squamous pearls, streaming	Streaming
Secretory/ glandular (adenocarcinoma)	Eccentric	Oval	Vacuoles	Fine	Present	Columnar	Acini (glandular structures), (true) papillae
Neuroendocrine	Center (?)	Oval	Fine granules (neuroendocrine granules - seen on Romanowsky type stains)	Reticulated "stippled"	Absent	Perinuclear blue bodies (not seen in lymphoid cells)	Clusters, may appear dyscohesive
Lymphoid	Eccentric	Oval	Very scant, basophilic	Coarse	Usually absent	Lymphoglandular bodies, dyscohesive	Single cells
Germ cell	Central/variable	Oval	Variable	Variable	(?), present in seminoma	Tigroid background in seminoma (Romanowsky stains)	Usu. in clusters
Mesenchymal	Central	Spindle	(?)	Fine (?)	Absent	Variable	Clusters, streaming

Vacuoles

Hydropic vacuoles and secretory vacuoles:

	Contents	Distorts nucleus	Significance
Hydropic vacuole	Empty (clear)	Less common	Dying cell
Secretory vacuole	"Globs" of stuff (usu. pink)	More common	Secretory cell/consider adenocarcinoma

Notes:

• Size the vacuole is not discriminative.
• Nuclear distortion (by the vacuole) is probably the strongest indicator the vacuole is secretory.

Subtyping adenocarcinoma

Adenocarcinoma can be subtyped:

	Architecture	Group borders	Group cellular/nuclear pleomorphism	Vacuoles (cytoplasmic)	Nucleoli	Other
Serous carcinoma	Micropapillae	Knobby	Marked size variation, +/-second benign pop.	Hydropic	Large, red	Psammoma bodies
Gastric (signet ring) carcinoma	Single cells, groups	Community border (?)	Limited, cells uniform, +/-second benign pop.	Mucin	Usu. inconspicuous	-
Breast ductal carcinoma	Clusters (tubular)	Community border	Limited, cells uniform, +/-second benign pop.	Mucin	Inconspicuous/small	-
Adenocarcinoma not otherwise specified (NOS)	3-D clusters	Community border	Limited, malignant cells uniform, +/-second benign pop.	Mucin	Inconspicuous/small	-
Mesothelioma (see Note 1)	Clusters (usu. large), single cells	Knobby border	Usu. uniform size, one cell population	Hydropic	Often large	Intercellular windows, cytoplasmic blebs

Note 1:

• Mesothelioma is a type of carcinoma, yet is not an adenocarcinoma; it is added to the table to make the point that it is similar to adenocarcinoma.
  • Mesothelioma arises from the mesothelium, a type of epithelium. Carcinomas are epithelial derived malignant tumours (by definition).

Staining

There are two standard stains:

1. Papanicolaou stain (Pap stain).
   • Is a modified H&E stain.
   • Done on alcohol-fixed material.
2. Romanowsky stain.
   • Many variants exist.
   • Used on air-dried specimens; air-dried cells are much larger, it is easier to see the nuclear detail.

Processing:

• Carnoy's solution (ethanol, glacial acetic acid) - used to lyze RBCs to get rid of 'em.

Further details are in the main article on staining.

Notes:

• Jörundsson et al. compare the types of stains.
• Romanowsky type staining is uncommon in the Canadian cytopathology community and not on FRCPC exams.

Pap stain

Interpretation:

• Blue = nucleus, basophilic cytoplasm.
• Pink = cytoplasm.
• Orange = keratin.

DDx of orange:

1. Keratin.
2. Dying/dead cell.
3. Center of cluster effect/diffusion effect.
   • The center of large clumps of cells are often artifactually pink/orange, as the eosin (a small molecule) get there but the azure (a large molecule) does not.

Romanowsky stains

Variants:

• Diff-Quik.
• Field's stain.
• May-Grünwald-Giemsa.
• Wright stain.

Interpretation (Romanowsky stain):

• Red - RBCs, eosinophil granules, neuroendocrine granules.
• Blue (basophilic) - lymphocyte cytoplasm.
• Purple - nuclear chromatin, neutrophil granules, platelets.

Notes:

• May have air-drying artifact.
  • Usually on slide periphery.
  • Appearance: white holes.
    • Air-drying may mimic the appearance of nuclear pseudoinclusions.
• Cells tend to be larger than on Pap stain.

Adequacy of specimens

Standards are lacking for most types of specimens. A generally accepted standard is established only for cervical cytology specimens.

Standards (UHN) for non-gyne specimens:

• Breast: >= 6 cell groups (6 cells/cluster).
• Thyroid: >= 20 cell cluster (20 cells/cluster).
• Urine: >=50 urothelial cells.
• Sputum: >= 10 pulmonary macrophages.

There is a generally accepted standard for cervical (liquid-based) cytology specimens:

• >5000 squamous cells/slide, if no abnormality is present.
  • If abnormal cells are present, any number of cells is acceptable.
    • This works-out to approx. 4 cells/HPF.
      • Where: HPF = area seen at 400X with an eye piece diameter is ~22 mm.
    • 10 HPFs are counted and a table is used to see whether the sample is adequate.

Note:

• The standard for conventional pap smears is: 8000-12000 squamous cells/slide.

Transformation zone (TZ)

The presence of the TZ should be commented on:

• An adequate TZ is 10 cells - endocervical cells or squamous metaplastic cells (per Bethesda).

Difficulties in obtaining a TZ may arise in the following populations:

• Pregnant (endocervical canal not sampled).
• Menopausal.
• Young nulliparous.

Principles of FNA

General:

• Fine needle aspiration is an oxymoron.
  • One does not really aspirate.

Principles:

• Use a small needle - 22 gauge.
• Use minimal suction - aspiration per se is not the key; the sample is obtained by coring.
• Use minimal back and forth angulation - it destroys blood vessels

History:

• Popularized by the Swedes.

Triage of specimens

At UHN the specimens are triaged. This is an uncommon practise in the world of cytopathology.

Options:

• Smear.
• Fancy (automated) thin layer preparations:
  • Cytospin - centrifuged onto slide.
  • ThinPrep - centrifuged onto filter then onto slide.
• Smear + IHC.
• Cell block. + IHC.

Triage of cytopathology specimens:

Triage of specimens

Morphologic analysis

Abnormal

Normal

Lymphoid

Non-lymphoid

Unknown

Rest to morphology

Management (liquid):

• Lymphoid:
  • Fresh:
    • Flow cytometry (FC) or laser scanning cytometry (LSC) if sufficient.
    • Additional preps (i.e. CytoSpin) not usually useful.
  • Fixed:
    • Minimal material: smear + immunostains.
    • Abundant material: cell block + immunostains.
• Non-lymphoid:
  • Cell block immunostains.
• Unknown:
  • Minimal material: smear + immunostains, possibly cell block + immunostains.
  • Abundant material: cell block + immunostains and flow cytometry.

Management (solid/clot):

• Cell block +/- immunostains.

Notes:

• It may be hard to diagnose large B cell lymphoma from LSC; thus, a cell block is useful if you have the material.
• Abundant macrophages cause problems with LSC.
• It is not worthwhile to do LSC if there is a scant amount of lymphoid tissue present in the specimen.
• LSC is (usually) not done on CSF specimens with less than 7 mL of volume.

Number of cells for LSC - calculation

$\psi _{20xo}V_{sample}={\frac {S_{needed}V_{RA}A_{20xo}}{A_{RA}}}$

Parameters

$S_{needed}=50,000~cells$ -- number of cells needed.
$V_{sample}=2-10~ml$ -- volume of sample.
$V_{RA}=0.5~ml$ -- est. volume of rapid assessment (RA).
$A_{RA}={\frac {\pi }{4}}(11)^{2}~mm^{2}$ -- est. rapid assessment (RA) area.
$A_{20xo}={\frac {\pi }{4}}(18/20)^{2}~mm^{2}$ -- area with 20x obj. on microscope with 18 mm eye piece.

Rule

$\psi _{20xo}V_{sample}=167.36~cells~ml$

Application of rule

1. Count cells in one field (on the microscope with the 18 mm eye piece) at 20x.
2. Divide 167.36 cells ml by the volume of the sample.
3. If the cell count in (1) is greater than the number of cells in (2) there are enough cells for a full panel.

Prepartions

	Cost	Cells required	Fixation	Disadvantages/problems
Smear	Minimal ~$5/slide	1+	Air dry/alcohol	Layering of cells
CytoSpin	Expensive	50	?	Expensive
ThinPrep (liquid-based cytology)	Expensive ~$35/slide; intermediate for several slides	300-400	Always alcohol (methanol)	Filter damage at edge of prep.
Sure-Path (liquid-based cytology) - see Note 1	Expensive (?)	300-400 (?)	Always alcohol (ethanol)	(?)
Cell block	Expensive	>500	Usually formulin	Expensive, loss of cells large, truncation artifact in FISH
Laser scanning cytometry	Expensive	~50,000 for full B cell panel + minimal T cell panel - see Note 2	None	Tissue needs to be fresh

Note 1:

• Sure-Path (from TriPath) is not used at many teaching centres as it has some short coming for non-gyne specimens vis-a-vis ThinPrep (from Cytyc).
  • It is used by the private labs in Ontario and seems to be better for Pap tests.
• A comparison between SurePath and ThinPrep/experience from one lab in the USA is here: http://www.palpath.com/MedicalTestPages/papthin1.htm.

Note 2 - see lymphoma article for detailed description of markers:

• The typical "full" panel is:
  • B-cell markers: CD10, CD19, CD20, CD23, FMC7, Kappa, Lambda.
  • T-Cell markers: CD3, CD5, CD4, CD8, CD7, CD2.
  • NK-Cell markers: CD56, CD16.
  • Miscellaneous markers: CD11c, CD14.

Notes (general):

• CytoSpin used for CSF specimens.

Cytologic features

Large clusters

• Large clusters - fibrin (junk) vs. other:
  • Small (~1/10 of RBC diameter) ovoid, purple staining (on Field stain) = platelets.
    • If you see platelets think fibrin.

Necrosis

• Blobs of acellular material.
• No nucleus or pyknosis of nucleus.
• Disrupted/frayed cellular membrane.
• Orangeophilic material (orange material) - on Pap stained material.

Granulomata

Histology (six features):

1. Foamy/bubbly cytoplasm, abundant - low power.
2. Epithelioid morphology - cell borders near indistinct - key feature.
3. "Footprint" pattern nuclei/bean-shaped nuclei - key feature.
   • Macrophages usu. have an ovoid nucleus.
4. +/-Nucleoli, small.
5. +/-Fibrosis.
6. +/-Pallisading at edge.

Notes:

• Granulomas are not always nice and round, as in non-cytology specimens.

Images:

• Granuloma - cytology (WC).
• Granuloma (pathconsultddx.com).
• Granuloma (cytology & core/surgical).

DDx:

• Infectious:
  • AFB:
    • Tuberculosis.
    • MAC.
  • Fungal:
    • Histoplasma.
  • Bacterial:
    • Bartonella (gram -ve) (Cat-scratch disease).

Detail articles

Details are dealt with in separate articles.

Gynecologic cytopathology

Main article: Gynecologic cytopathology

Gynecologic cytopathology is the origins of cytopathology and still a large part of cytopathology practise. It is deal with in separate article.

CNS cytopathology

Main article: CNS cytopathology

The above article deals with CNS cytopathology, i.e. lumbar puncture specimens.

Pulmonary cytopathology

Main article: Pulmonary cytopathology

The above article deals with pulmonary cytopathology, i.e. FNA specimens and sputum specimens.

Fluid from mesothelial-lined space cytopathology

Main article: Mesothelial cytopathology

The above article deals with cytopathology specimens from spaces lined with mesothelium.

Urine cytopathology

Main article: Urine cytopathology

The above article deals with urine cytopathology.

Thyroid cytopathology

Main article: Thyroid cytopathology

The above article deals with thyroid cytopathology.

Head & neck cytopathology

Main article: Head and neck cytopathology

The above article deals with head and neck cytopathology, excluding thyroid cytopathology.

Breast cytopathology

Main article: Breast cytopathology

The above article deals with breast cytopathology.

Grab bag of stuff

The following is a grab bag of stuff that is:

• Not covered in the above articles.
• Something that applies to multiple sites (e.g. melanoma).

Mediastinum

DDx:

• Teratoma and other germ cell tumours (GCTs).
  • Seminoma/dysgerminoma is the most common.
• Terrible lymphoma.
• Thymoma.
  • Mix of epithelial cells and small lymphoid cells.
• Thyroid.
• Metastasis.

Thymoma

Cytopathology

Features:

• Mixed population of cells:
  • Epithelial cells.
    • Circular nuclei with well seen micronucleoli (seen at ~10X objective).
    • +/-Nuclear membrane invaginations.
  • Mature Lymphocytes.

DDx (pitfalls):

• Lymphoproliferative disorder (when epithelial component sparse).
• Carcinoma (when lymphoid population small).
  • Should not have nuclear features of malignancy.
    • Adenoca.: marked size variation, larger nucleoli.
    • Squamous ca.: often no nucleoli. (???)

Notes:

• May have necrosis.
• Should not mistake for granuloma... as nuclei of epithelial cells round not oval.

Image:

• Thymoma - Field stain (WC).

Germ cell tumours

Seminoma

Features:

• +/-"Corners" in the nuclear membrane.
• Cells cohesive.
• Central nucleus.
• Clear cytoplasm.
• Background with "viscous" or "tigroid" appearance on (air dried) Romanowsky type stains - key feature.
  • Tigroid = striped or spotted appearance.
    • Related to the presence of glycogen.
  • Tigroid pattern not seen on Pap stain.
  • Not always seen.
• +/-Small mature lymphocytes (common).
• +/-Granulomas.

Gastrointestinal

Liver

Normal:

• Liver cells may be binucleated.
• Hepatocytes:
  • Abundant cytoplasm, central nucleus.
  • Bile ductules between adjacent cells.
  • +/-Yellow granular pigment (bile).

Hepatocellular carcinoma:

• Clusters of liver cells surrounded by endothelial cells.
• +/-Nuclear atypia.

Notes:

• Low grade HCC is composed of cytologically normal appearing cells; the arrangement is what is diagnostic of malignancy.

Cholangiocarcinoma:

• Looks like an adenocarcinoma:
  • Eccentric nuclei, one nucleolus per cell, abundant cytoplasm, nuclear size var. cell-to-cell, irregular nuclear membrane, irregular/uneven chromatin pattern.

Common bile duct

Normal:

• Monolayer of small blue cells.

Notes:

• Caution is advised when calling malignancy in the setting of a stent or stones.

Adenocarcinoma

Features:

• Hyperchromasia.
• Pencil-shaped nuclei.
• Nuclear membrane irregularities.

Images:

• Colorectal adenocarcinoma - low mag. (WC).
• Colorectal adenocarcinoma - intermediate mag. (WC).

Stomach

• May be difficult to distinguish from pancreas ductal epithelium.

Small bowel

Epithelium:

• Small blue cells.
• Goblet cells - key feature.

Notes:

• May appear to be similar to stomach and pancreatic duct.

Esophagus

• Cytology may be done to look for candida.
  • Report should comment on the presence of candida - if it is seen.

Renal oncocytoma

Features:

• Moderate granular cytoplasm.
• Distinct cell borders.
• Round nuclei.

Image:

• Renal oncoctyoma - resection (WC).

Oncocytoma vs chromophobe RCC:

• ChRCC:
  • Thicker cell borders.
  • Perinuclear clearing/quasi koilocytic appearance.

Crystals

Joint crystals

Types:

• Gout = needle-shaped, negatively birefringent (yellow).
• Pseudogout = rhomboid-shaped, postively birefringent (blue).

Notes:

• Pseudogout also known as CPPD = calcium pyrophosphate dehydrogenase.

Urine crystals

Types:

• Envelope shape (calcium oxalate).
• Diamond shape (uric acid).
• Coffin-lid shape (struvite).
• Hexagonal shape (cysteine).

Notes:

• Memory device: Diamonds are see-through; ergo, uric acid stones not seen on KUB.
• Uric acid crystals: usually dissolve in formulin... but do not dissolve in alcohol.

Skin

Malignant melanoma

Classic features:

1. Loosely cohesive cells and single cells.
2. Mixure of epithelioid cells and spindle cells.
3. Malignant cells have:
   • Prominent red nucleolus.
   • Pigmented cytoplasm - key feature (often not pigmented).
   • Pigment may only be present in macrophages
   • Occasional large binucleated cells (bug-eyed monster cell).
     • Nuclei are often at opposite poles of the cell, i.e. the nuclei are as far apart as possible ("divorce cells").
   • Intranuclear inclusions.
4. Pigmented macrophages (useful feature - but less specific for melanoma than pigment in malignant looking cells).

Notes:

1. Large nucleolus - may Vaguely resemble adenocarcinoma.
   • Prominent red nucleolus common in: serous carcinoma.
2. The classic appearance of melanoma without pigment is closest to adenocarcinoma (which may have red nucleoli, large cells, abundant cytoplasm, occasional binucleation).
   • Differentiating morphologic features: adenocarcinoma - 3-D clusters of cells, no spindle-shaped cells.
3. Bug-eyed monster cells - may vaguely resemble a Reed-Sternberg cell (RSC) - diagnostic of Hodgkin's lymphoma (HL).
   • RSCs do not have the granular cytoplasm typical of melanoma.
   • Nuclei usually adjacent, i.e. not at opposite poles of the cell.
   • Background of melanoma different than HL.

Images:

• Bug-eyed monster cell in melanoma - Field stain (WC).
• Pigmented melanoma - Pap stain (WC).

Bone marrow/vertebrae

Components:

1. One should see three lineages:
   • Megakaryocytes.
     • Large cells ~ 5-10X RBC.
     • Eccentric nuclei.
   • Erythrocyte precursor.
     • Central nucleus.
     • Chromatin - granular.
     • One should see ejected nuclei of the same size.
   • Granulocytes.
2. Fat.
   • May be seen in a lymph node.

Notes:

• Megakaryocytes - reduced post-bone marrow transplant.

Short DDx:

• Plasma cell lesion, e.g. plasmacytoma.
• Carcinoma (metastatic).
• Lymphoma.

See also

• Basics.

External links

• Cytopathology Institute Duesseldorf.
• Cytopathology test (jhu.edu).
• BC cancer agency quiz (bccancer.bc.ca).